Cytotoxic factors for modulating cell death

ABSTRACT

Cytotoxic factors having use in modulating cell death, and their use in methods of treating necrosis or apoptosis-related conditions are disclosed. The invention also relates to methods for identifying active agents useful in treating conditions related to cell death or uncontrolled growth. The present inventors have found that different microorganisms produce different cytotoxic factor(s) having anticancer activity. The substantially pure cytotoxic factors can be used in a method of treating an infectious disease or a cancer.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/414,550 filed Aug. 15, 2003, and is a continuation-in-partof U.S. patent application Ser. No. 10/047,710, filed Jan. 15, 2002 nowU.S. Pat. No. 7,084,105, which claims priority to U.S. ProvisionalPatent Application Ser. No. 60/269,133, filed Feb. 15, 2001. The entirecontent of these prior applications is fully incorporated herein by thisreference.

STATEMENT OF GOVERNMENTAL INTEREST

The subject matter of this application has been supported by researchgrants from the National Institutes of Health (NIH), Bethesda, Md.,U.S.A., (Grant Numbers AI 16790-21, ES 04050-16, AI 45541, CA09432 andN01-CM97567). The government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to cytotoxic factors secreted bymicroorganisms and inhibitors of cytotoxic factors and their use incausing cellular growth arrest and in modulating cell death by necrosisand apoptosis. The present invention also relates to methods ofproducing, isolating and identifying such cytotoxic factors and tocompositions incorporating substantially pure cytotoxic factors usefulin modulating cell death and causing cellular growth arrest. Theinvention also relates to methods of treating apoptosis-relatedconditions. More particularly, the invention relates to the use of asubstantially pure cytotoxic factor in a method of inducing apoptosis orcellular growth arrest in a cancer cell and to the use of inhibitors ofthe cytotoxic factors for treating an infection or otherpathogen-induced condition.

BACKGROUND

Infectious diseases can be a product of a number of environmentalfactors. Underlying any infectious disease is a causative infectiousagent. The infectious agent typically is a pathogenic microorganism, forexample, a pathogenic bacterium. The degree or ability of a pathogenicmicroorganism to overcome defense mechanisms and cause a disease isrelated to its virulence. Both pathogenic and nonpathogenicmicroorganisms are known to express cytotoxic factors, which allow themicroorganism to defend itself from the host immune system and preventphagocytes (e.g., macrophages and mast cells) from eliminating themicroorganism from the body. When the pathogenic microorganisms survive,the microorganisms can invade the host tissues and proliferate, causingsevere disease symptoms. Pathogenic bacteria have been identified as aroot cause of a variety of debilitating or fatal diseases including, forexample, tuberculosis, cholera, whooping cough, plague, and the like. Totreat such severe infections, drugs, for example, antibiotics, areadministered that either kill the infectious agent or disarm thecytotoxic factors so that the infectious agent is no longer able todefend itself against the host immune system. However, pathogenicbacteria commonly develop resistance to antibiotics and improved agentsare needed to prevent the spread of infections due to suchmicroorganisms.

A cancer is a malignant tumor of potentially unlimited growth. It isprimarily the pathogenic replication (a loss of normal regulatorycontrol) of various types of cells found in the human body. Initialtreatment of the disease is often surgery, radiation treatment or thecombination of these treatments, but locally recurrent and metastaticdisease is frequent. Chemotherapeutic treatments for some cancers areavailable but these seldom induce long term regression. Hence, they areoften not curative. Commonly, tumors and their metastases becomerefractory to chemotherapy, in an event known as the development ofmultidrug resistance. In many cases, tumors are inherently resistant tosome classes of chemotherapeutic agents. In addition, such treatmentsthreaten noncancerous cells, are stressful to the human body, andproduce many side effects. Improved agents are therefore needed toprevent the spread of cancer cells.

Many cancers are known to regress when patients are infected withpathogenic bacteria. However, very little is known about how bacterialinfections cause regression of human cancers.

SUMMARY

The present invention relates to cytotoxic factors that stimulate celldeath by necrosis or apoptosis or that cause cellular growth arrest. Inone aspect, substantially pure cytotoxic factors have been characterizedand isolated. Substantially pure cytotoxic factors are obtained bycolumn chromatographic fractionation of a growth medium which has beenexposed to a pathogenic microorganism. Preferably, the production andsecretion of such cytotoxic factors are stimulated during growth ofpathogenic organisms in the presence of mammalian proteins.

In another aspect of the present invention, the identification ofreceptors for mammalian proteins as a means of delineating virulent andavirulent microorganisms can lead to improved specificity for diseasetreatment.

Yet another aspect of the present invention relates to a method oftreating a condition related to cell death resistance or susceptibilitycomprising the step of administering a cytotoxic factor, an inhibitor ofa cytotoxic factor, or a variant or derivative thereof, optionallyincorporated in a pharmaceutical carrier.

The cytotoxic factor, or a variant or derivative thereof, can beincorporated into a pharmaceutical composition for use in the preventionand treatment of conditions related to abnormal cell proliferation. Forexample, a cytotoxic factor can be used to treat a cancer.

An inhibitor of a cytotoxic factor, or a variant or derivative thereof,can be used to treat a bacterial infection by preventing phagocytic celldeath and hence allowing the host immune system to combat an invadingpathogen.

In another embodiment of the present invention, cytotoxic factors, aswell as components of their secretion machinery, can be used ascandidates for vaccines against infectious agents.

The present invention also relates to a method of modulating cell deathcomprising the step of controlling secretion of cytotoxic factors. Inone embodiment, the cytotoxic factors can be used as anti-cancer agentsagainst a host of human cancer cells. Cytotoxic factors can also be usedas targets for drug development through screening or rational design ofinhibitors.

The present invention also relates to a method of modulating cell deathcomprising utilizing a cytotoxic factor such as an azurin, aplastocyanin, a rusticyanin, a pseudoazurin, or a cytochrome c551, or amutant of such a cytotoxic factor.

These and other aspects, advantages, and features of the invention willbecome apparent from the following figures and detailed description ofthe preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chart showing the effect of 1.0 mM ATP on macrophage killing inabsence or in presence of the filtered growth medium supernatant (SUP)or the hydroxyapatite flow through (HAFT), ATP-agarose flow through(AAFT) and Q-sepharose flow through (QSFT) column chromatographicfractions derived from B. cepacia growth medium. The extent ofmacrophage cell death is measured by release of the intracellular enzymelactate dehydrogenase (LDH). 2 μg of protein from each fraction was usedin the assay. All assays were carried out in triplicate and error barsare indicated.

FIG. 2. Chart showing the effect of filtered growth medium supernatant(SUP) and column chromatographic fractions (HAFT, AAFT and QSFT) of B.cepacia on macrophage cell death in the absence of ATP. The extent ofmacrophage cell death is measured by the release of the intracellularenzyme lactate dehydrogenase (LDH). All assays were carried out intriplicate and error bars are indicated.

FIG. 3. Graphs showing caspase activities (FIG. 3A—caspase-3; FIG.3B—caspase-9) in the cytosolic extracts of J774 macrophages treated withB. cepacia QSFT fraction. Cytosolic extracts were prepared frommacrophages incubated overnight with B. cepacia QSFT fraction (10 μgprotein) and from untreated macrophages. The substrate for thedetermination of caspase-3 activity was Ac-DEVD-pNA(N-acetyl-Asp-Glu-Val-Asp-p-NO₂-aniline). The substrate for caspase-9activity was Ac-LEHD-pNA (N-acetyl-Leu-Glu-His-Asp-p-NO₂-aniline).Extracts were incubated with the substrate at 37° C. for the timesindicated. 10 μg of macrophage cytosolic protein was used in each case.Release of pNA (p-nitroaniline) was determined spectrophotometrically at405 nm.

FIG. 4. Chart showing cytotoxicity, as measured by % lactatedehydrogenase (LDH) release, in macrophages in presence of azurin (Az),cytochrome C₅₅₁ (Cyt C₅₅₁) and combination thereof. The numbersrepresent μg protein. The buffer control (buffer) is shown at right.

FIG. 5. Chart showing the effects of anti-azurin and anti-cytochromec551 antibodies on cytotoxicity of B. cepacia (A) and M. bovis (B) QSFTfractions and in the presence of preimmune serum. A, azurin (50 μg); C,cytochrome c551 (25 μg); ab, combination of anti-azurin andanti-cytochrome c551 antibodies; P, preimmune serum. 2 μg of QSFTfraction were used in each assay. The numbers after ab and P representμg of the antibody or preimmune protein. Results shown aremeans±standard deviations of triplicate experiments.

FIG. 6. Graph showing the effect of post injection of azurin/cytochromec₅₅₁ in nude mice on the size of the tumor after induction of melanomatumor cells (UISO-Mel-2). Approximately 10⁶ UISO-Mel-2 cells wereinjected subcutaneously in nude mice followed by once weeklyintraperitoneal injections of either citrate buffer (control), a knownanti-melanoma drug DTIC (7.5 μg) or three times per week a high (150 μgazurin/75 μg cytochrome c₅₅₁) or low (10 μg azurin/5 μg cytochrome c₅₅₁)dose of azurin/cytochrome c₅₅₁ mixture for 4 weeks. At various times,the sizes (tumor volume) of the tumors in control (buffer treated),DTIC-treated and high and low dose azurin/cytochrome c₅₅₁-treated micewere determined and plotted graphically.

FIG. 7. Graph showing gain or loss of weight of the mice during theexperiment described under FIG. 6. During the course of the aboveexperiment, the mice were weighed on a scale and the weights in gramsnoted.

FIG. 8. Graph showing regression of Mel-6 tumor in nude mice treatedwith M. Bovis QSFT fraction in the presence or absence of azurin (AZ).Approximately 10⁶ UISO-Mel-6 cells were injected subcutaneously in nudemice. Small tumors developed after approximately one week. The mice werethen intraperitonealy injected with phosphate buffered saline (control),M. Bovis QSFT fraction or a mixture of M. Bovis QSFT fraction andazurin.

FIG. 9. Graph showing the cytotoxicity of azurin for MCF-7 (▪) andMDA-MB-157 (□) cells treated with various concentrations of azurin for72 hours.

FIG. 10. Graph showing regression of MCF-7 tumor in nude mice treatedwith azurin (▪) and control animals (□).

FIGS. 11( a) and (b). FIG. 11( a) is a table showing the alignment ofthe amino acid sequence of PA, P. aeruginosa azurin (SEQ ID NO: 23),with other bacterial azurins; AF, Alcaligenes faecalis (SEQ ID NO: 24);AX, Achromobacter xylosoxidans ssp. denitrificans I (SEQ ID NO: 25); BB,Bordetella bronchiseptica (SEQ ID NO: 26); MJ, Methylomonas sp. J (SEQID NO: 27); NM, Neisseria meningitidis Z2491 (SEQ ID NO: 28); PF,Pseudomonas fluorescen (SEQ ID NO: 29); PC, Pseudomonas chlororaphis(SEQ ID NO: 30); XE, Xylella fastidiosa 9a5c (SEQ ID NO: 31). Amino acidsequences are aligned by Genetyx software. FIG. 11( b) is a tableshowing wild type azurin (wt azurin, SEQ ID NO: 44) and chimeric mutantazurins; S1 (SEQ ID NO: 45); S2 (SEQ ID NO: 46); S3 (SEQ ID NO: 47);S3S5(SEQ ID NO: 48); S3S5S4S6(SEQ ID NO: 49); S4 (SEQ ID NO: 50); S6(SEQ ID NO: 51); WtS5 (SEQ ID NO: 52); and WtS5S4S6(SEQ ID NO: 53).

FIGS. 12( a) and 12(b). FIG. 12( a) is a graph showing the cytotoxicityof wild type and redox mutant azurins towards macrophage cells. Wildtype azurin (●), apo-azurin (∘), M44KM64E (▴), C112D (Δ).

FIG. 12( b) is a graph showing the cytotoxity of wild type and chimericmutant azurins towards macrophage cells. Wild type azurin (●), S1 (∘),S2 (▴), S3 (▪), S4 (Δ), S6 (□), wtS5 (▾), wtS5S4S6 (♦), S3S5 (∇). FIG.12( b) also shows the relative electron transfer efficiency of themutants expressed as a percentage of that of wild type azurin. Tocalculate percentage cytotoxity, the number of non-treated viable cellswas taken as 100% and the number of viable cells in the azurin-treatedsamples determined.

FIG. 13 is a graph showing the apoptotic activity of azurin, apo-azurin,and azurin mutants towards macrophage cells. Wild type azurin (●),apo-azurin (∘), M44KM64E (▴), C112D (Δ).

FIG. 14 is a graph showing the cytotoxicity of wild type azurin (wtazu▴), rusticyanin (crude●), apo-rusticyanin (apo-rus ∘), andpseudoazurin (Paz □).

FIG. 15 is a graph showing the cytotoxicity of wild type azurin (●) andplastocyanin (▪).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Definitions

For the purposes of the description herein, the term “cytotoxic factor”refers to a factor secreted by a pathogenic or nonpathogenicmicroorganism and that stimulates cell death by necrosis or apoptosis orthat causes cellular growth arrest. Examples of cytotoxic factorsinclude an azurin, a plastocyanin, a rusticyanin, a pseudoazurin, or acytochrome c551. The term “ATP-dependent”, when used to modify the term“cytotoxic factor” refers to a cytotoxic factor which acts to cause celldeath or cellular growth arrest in the presence of adenosine5′-triphosphate (ATP). The term “ATP-independent”, when used to modifythe term “cytotoxic factor” refers to a cytotoxic factor which acts tocause cell death or cellular growth arrest in the absence of ATP.

For the purposes of the description herein, the term “treatment”includes preventing, lowering, stopping, or reversing the progression orseverity of the condition or symptoms being treated. As such, the term“treatment” includes medical, therapeutic, and/or prophylacticadministration, as appropriate.

As used herein, the term “a condition related to resistance to celldeath” refers to a disease, state, or ailment characterized by at leasta tendency for prolonged cell life when compared with a healthy cell oflike kind as determined by a reasonable, skilled physician or clinician.The term “a condition related to cell death susceptibility”, as usedherein, refers to a disease, state, or ailment characterized by at leasta tendency for premature cell death when compared with a healthy cell oflike kind as determined by a reasonable, skilled physician or clinician.

As used herein, the term “having a functional p53 tumor suppressor gene”refers to a cell having a p53 tumor suppressor gene that is notinactivated, mutated, lost or under produced.

As used herein, the term “deficient in p53 tumor suppressor gene” refersto a cell having a p53 tumor suppressor gene that is inactivated,mutated, lost or under produced. For example, such a deficiency mayoccur as a result of genetic aberrations within the p53 gene orinteraction with viral and cellular oncogenes.

The term “substantially pure”, when used to modify the term “cytotoxicfactor”, as used herein, refers to a cytotoxic factor, for example, acytotoxic factor isolated from the secreted growth medium, in a formsubstantially free of, or unadulterated by, active inhibitory compounds.The term “substantially pure” refers to a factor in an amount of atleast about 75%, by weight, of isolated fraction, or at least “75%substantially pure”. More preferably, the term “substantially pure”refers to a compound of at least about 85%, by weight, active compound,or at least “85% substantially pure”. The substantially pure cytotoxicfactor can be used in combination with one or more other substantiallypure compounds or isolated cytotoxic factors.

As used herein, the term “a variant or derivative” of a cytotoxic factorrefers to a compound or substance obtained by chemical modification ormanipulation of the cytotoxic factor or the gene encoding the cytotoxicfactor. The variant or derivative of a cytotoxic factor can be obtainedby chemical modification of the cytotoxic factor, or by manipulation ofgenes encoding the cytotoxic factor, for example by altering the basiccomposition or characteristics of the cytotoxic factor, but not itstoxicity. Similarly, “a variant or derivative” of an inhibitor of acytotoxic factor can include chemical modifications to the chemicalstructure of the inhibitor or manipulation of genes encoding theinhibitor.

The term “percent (%) amino acid sequence identity” is defined as thepercentage of amino acid residues in a cytotoxic factor that areidentical with amino acid residues in a candidate sequence when the twosequences are aligned. To determine % amino acid identity, sequences arealigned and if necessary, gaps are introduced to achieve the maximum %sequence identity; conservative substitutions are not considered as partof the sequence identity. Amino acid sequence alignment procedures todetermine percent identity are well known to those of skill in the art.Often publicly available computer software such as BLAST, BLAST2, ALIGN2or Megalign (DNASTAR) software is used to align peptide sequences.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=X/Y·100

where

X is the number of amino acid residues scored as identical matches bythe sequence alignment program's or algorithm's alignment of A and B and

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A.

A “therapeutically effective amount” is an amount effective to preventdevelopment of, or to alleviate the existing symptoms of, the subjectbeing treated. Determination of a therapeutically effective amount iswell within the capability of those skilled in the art.

General

The present invention provides cytotoxic factors that are secreted bypathogenic or nonpathogenic microorganisms and that stimulate cell deathby necrosis or apoptosis or that cause cellular growth arrest. Whenpathogenic microorganisms invade human or animal tissues, phagocyticcells are a first line of defense in the host immune system. Typically,phagocytes seek out and destroy foreign pathogens invading the body.However, cytotoxic factors secreted by microbial pathogens can stimulatecell death in the phagocytic cells. Thus, the phagocytes are preventedfrom performing their protective immune function.

The inventors have previously reported that many pathogenic bacteriasecrete ATP-dependent cytotoxic factors, for example ATP-utilizingenzymes, that cause phagocytic cell death by necrosis. [Zaborina O. etal., Infect. Immun. 67: 5231-5242 (1999); Melnikov A. et al., Mol.Microbiol. 36: 1481-1493 (2000); and Punj V. et al., Infect. Immun. 68:4930-4937 (2000), the contents of which are incorporated for allpurposes by the reference.] ATP-utilizing enzymes act on variousenergy-related nucleotide derivatives such as ATP, adenosine5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), or adenosine,converting them to various products that in turn can modulate the deathof phagocytic cells such as macrophages and mast cells throughactivation of purinergic receptors.

One aspect of the present invention relates to the discovery thatATP-independent cytotoxic factors, for example redox proteins, are alsosecreted by some species of pathogenic microorganisms, and that suchfactors cause phagocytic cell death by apoptosis. [Zaborina O. et al.,Microbiology 146: 2521-2530 (2000), the contents of which areincorporated for all purposes by the reference.]

Another aspect of the present invention relates to the surprisingdiscovery that ATP-independent cytotoxic factors induce apoptosis orcellular growth arrest in cancer cells. Such cytotoxic factors may beused to treat a condition related to resistance to cell death. Suchconditions may include, for example, human melanoma, leukemia, breastcancer, ovarian cancer, lung cancer, mesenchymal cancer, colon cancerand aerodigestive tract cancers (e.g. stomach, esophagus, larynx andoral cancers).

Normally cancer cells are not susceptible to apoptotic death. Suchresistance to cell apoptotic cell death can be caused by inactivatingmutations in the gene encoding the p53 tumor suppressor protein. It isknown that mammalian cell apoptosis requires the presence of p53protein. However, in 50% of human cancers, inactivating mutations in thegene encoding the p53 tumor suppressor protein are present.

Although it is also known that p53 regulates the expression of redoxproteins in mammalian cells, mammalian redox proteins have not beendirectly implicated in cancer cell apoptosis or growth arrest. Neitherhas the role of microbial ATP-independent cytotoxic factors in inducingapoptosis in cancer cells or in reducing tumor size been shown.

Another aspect of the present invention relates to methods ofidentification and characterization of cytotoxic factors secreted bymicroorganisms. Such methods can provide a means for discoveringappropriate inhibitors or stimulators of cell death. Inhibitors andstimulators can be developed as pharmaceutical drugs and used to treatconditions characterized by resistance or susceptibility to cell death.

Another aspect of the invention relates to cytotoxic factors that havebeen characterized and isolated and to inhibitors of such cytotoxicfactors. The cytotoxic factors can be activated or inactivated inaccordance with a method of the invention to prevent or treat acondition related to cell death. An inhibitor of a cytotoxic factor canbe used to treat a condition related to cell death susceptibility.

Secretion of Cytotoxic Factors

In one aspect of the present invention, cytotoxic factors of the presentinvention are secreted by a number of different pathogenicmicroorganisms, including bacteria and protozoa. Examples of pathogenicbacteria suitable for providing the cytotoxic factors include, but arenot limited to, Pseudomonas aeruginosam (P. aeruginosa), Burkholderiacepacia (B. cepacia), Vibrio cholerae (V. cholerae), and Mycobacteriumbovis (M. bovis). In addition, cytotoxic factors are secreted bypathogens, such as Leishmania amazonensis and Brugia malayi.

P. aeruginosa, an opportunistic pathogen, B. cepacia, which causes fatalinfections in patients suffering from cystic fibrosis and chronicgranulomatous disease, V. cholerae, the intestinal pathogen that causescholera and the slow-growing virulent group of mycobacteria, such as Mtuberculosis or M. bovis, that cause tuberculosis have been found tosecrete ATP-utilizing enzymes.

In addition to secreting ATP-utilizing enzymes, the inventors have foundthat P. aeruginosa secretes ATP-independent cytotoxic factors. Thesehave been identified as two redox proteins, azurin and cytochrome C₅₅₁ .B. cepacia has also been shown to secrete the redox proteins. M. bovishas been shown to also secrete cytotoxic factors having highATP-independent cytotoxicity towards phagocytic cells.

Stimulation of the Secretion of Cytotoxic Factors in the Presence ofMammalian Proteins

In another aspect of the present invention, production and secretion ofcytotoxic factors are stimulated during growth of pathogenic organismsin the presence of mammalian proteins. For example, the secretion ofcytotoxic factors by pathogenic microorganisms such as P. aeruginosa, M.bovis and B. cepacia is stimulated by the presence of mammalian proteinssuch as kappa-casein, bovine serum albumin, ovalbumin orα2-macroglobulin. It is suggested, but not relied upon herein, that thepathogenic microorganisms sense the presence of certain mammalianproteins as indicative of the mammalian host environment, therebyopening up the secretion machinery for the cytotoxic agents to counterand subvert host defense.

The inventors have determined that several clinical (virulent) isolatesof B. cepacia secrete large amounts of ATP-utilizing enzymes such asadenylate kinase or 5′-nucleotidase, while several environmental(avirulent) isolates secreted only reduced amounts of these enzymes. Inclinical isolates, such as B. cepacia strain 38, the level of secretionof cytotoxic factor is greatly enhanced in the presence ofα2-macroglobulin in the growth medium. The secreted products fromclinical isolates have a higher level of cytotoxicity towardsmacrophages and mast cells than that from environmental isolates. Theclinical isolates that demonstrate enhanced secretion of cytotoxicfactors in the presence of α2-macroglobulin also demonstrate thepresence of the receptors for α2-macroglobulin on their surface.

In one embodiment of the present invention, the production and secretionof ATP-independent cytotoxic factors are stimulated during growth ofmicroorganisms in the presence of mammalian proteins. Increasedsecretion of cytotoxic factors can be obtained by growing microorganismsorganisms in growth media containing mammalian proteins. Suitable growthmedia are, for example, L broth, nutrient broth, Trypticase soy brothand tryptone-yeast extract broth (Difco Laboratories, Maryland, U.S.A.).Typically, approximately 500 ml to 1,000 ml of sterile autoclaved growthmedium are inoculated with between about 10⁴ to 10⁶ cells/ml. Theinoculated medium is then incubated under conditions suitable to allowgrowth of the microorganism, usually on a rotary shaker at 30° C. to 37°C. Selection of growth media, incubation conditions, and other factorsallowing successful culture of bacteria and other microorganisms will beclearly apparent to one skilled in the art. The inventors have observedthat maximum concentrations of cytotoxic factors in the growth mediumoccur late in the exponential growth phase and early in the stationarygrowth phase.

In another embodiment of the present invention, the identification ofreceptors for mammalian proteins provides a means of delineatingvirulent and avirulent strains of microorganisms. For example, thepresence of the receptors for α2-macroglobulin primarily in clinicalisolates, but not in environmental isolates, not only correlates withthe ability of the former to secrete cytotoxic agents as weapons againstthe host defense, but also allows delineation between the clinical,virulent strains with the environmental, avirulent strains. Hence,virulent strains of organisms can be identified and then tested fortheir antibiotic sensitivity or for other clinical purposes.

Purification of ATP-Independent Cytotoxic Factors

In another aspect of the present invention, substantially pureATP-independent cytotoxic factors are obtained by column chromatographicfractionation of the growth medium of the secreting microorganism.Preferably, the bacterial cells are removed from the growth medium priorto fractionation. This may be achieved by initial centrifugation andsubsequent filtering the growth medium. Suitable filters are typicallyless than or equal to about 0.5 μm pore size and preferably about 0.2μm. However, other methods of pathogen removal will be known to thoseskilled in the art.

Unfractionated growth media do not have high ATP-independent cytotoxicactivity and hence column chromatographic fractionation is necessary toenhance apoptosis-inducing or cellular growth arresting activity.Fractionation removes ATP-dependent cytotoxic factors. It is alsosuggested, but not relied upon herein, that fractionation also removesinhibitors of ATP-independent cytotoxic factors that may be present inthe unfractionated growth medium.

Chromatographic techniques useful in purifying cytotoxic factors will beknown to those skilled in the art. These include, for example,ion-exchange chromatography, hydroxyapatite chromatography, affinitychromatography, and gel-filtration chromatography. Chromatographiccolumns useful in the fractionation of bacterial growth media include,for example: Hydroxyapatite; Superdex 75 or 200; Superose 6 or 12;Sephacryl S; Sephadex G or Sephadex LH; Mono Q or Mono S; Q-Sepharose;DEAE Sepharose or CM Sepharose; Sepharose XL; ATP-Sepharose; Hi TrapBlue; Blue Sepharose; DNA Cellulose or Sepharose 2B, 4B or 6B, availablefrom Amersham Pharmacia Biotech AB, Uppsala, Sweden or Bio-RadLaboratories, Hercules, Calif., U.S.A.

ATP-utilizing enzymes may be isolated by column chromatographicfractionation as flow-through or eluted fractions of hydroxyapatite andATP-agarose columns. During such fractionation, the ATP-utilizingenzymes, such as ATPase or adenylate kinase are adsorbed on the columnand can be removed or purified further. (See, for example, Markaryan etal., J. Bacteriol., 183, pp 3345-3352, 2001.)

In one embodiment of the present invention, ATP-independent cytotoxicfactors are isolated as flow-through fractions of Q-sepharose columns(QSFT). Q-sepharose is a quaternary ammonium strong anion exchanger.Such columns can be obtained from Amersham Pharmacia Biotech AB,Uppsala, Sweden. The supernatant (SUP) or other column fractions such ashydroxyapatite column flow through fraction (HAFT) or ATP-agarose columnflow through fraction (AAFT) do not normally show high ATP-independentcytotoxicity.

Characterization of ATP-Independent Cytotoxic Factors

In a further aspect of the present invention, fractionated growth mediaare tested to determine the presence of ATP-independent cytotoxicfactors. The extent of cell death may be measured by the release of theintracellular enzyme lactate dehydrogenase (LDH) as described inZaborina et al., Infection and Immunity, 67, 5231-5242 (1999) andZaborina et al., Microbiology, 146, 2521-2530 (2000), the contents ofwhich are incorporated for all purposes by this reference.

The ability of ATP-independent cytotoxic factors to induce apoptosis maybe observed by mitosensor ApoAlert confocal microscopy using aMITOSENSOR™ APOLERT™ Mitochondrial Membrane Sensor kit (ClontechLaboratories, Inc., Palo Alto, Calif., U.S.A.). In the assay, healthy,non-apoptotic cells fluoresce red while apoptotically dead cellsfluoresce green. A combination of red and green produces yellowfluorescing cells that represent apoptotically dying cells. See Zaborinaet al., Microbiology, 146, 2521-2530 (2000), the contents of which areincorporated for all purposes by this reference.

Apoptosis is mediated via activation of a cascade of enzymes known ascaspases, which are cysteine proteases cleaving at aspartic residues.Hence, apoptosis may also be detected by measuring two important caspaseactivities, namely that of caspase 9 and caspase-3, which are known tobe activated during apoptosis by the oligomerization of the cytochrome creleased from mitochondria with a cytosolic protein Apaf-1, using themethod described in Zou et al., J. Biol. Chem., 274: 11549-11556 (1999),the contents of which are incorporated for all purposes by thisreference.

Apoptosis may also be observed by detecting apoptosis-induced nuclearDNA fragmentation using, for example, the APOLERT DNA fragmentation kit(Clontech Laboratories, Inc., Palo Alto, Calif., U.S.A.). This assay isbased on terminal deoxynuclotidyltransferase (Tdt)—mediated dUTPnick-end labeling (TUNEL), where Tdt catalyzes the incorporation offluorescein-dUTP at the free 3′-hydroxyl ends of fragmented DNA in cellsundergoing apoptosis. The incorporation of fluorescein-dUTP in thefragmented nuclear DNA generates green fluorescence which is detected byconfocal microscopy.

In one embodiment of the present invention, fractionated growth mediaare tested to determine the ability of such fractions to induceapoptosis or cellular growth arrest. Such methods are useful in theidentification and characterization of ATP-independent cytotoxicfactors.

Identification of ATP-Independent Cytotoxic Factors

In another aspect, this invention provides characterized cytotoxicfactors exhibiting ATP-independent apoptosis-triggering cytotoxicity orthat cause cellular growth arrest. The inventors have found that theQSFT fraction of P. aeruginosa and B. cepacia is enriched with twoproteins, azurin and cytochrome C₅₅₁. The identification of these twoproteins is based on their separation on SDS-PAGE and identification oftheir N-terminal amino acid sequences. In contrast, SDS-PAGE analysis ofthe M. bovis QSFT fraction shows a thick 65 kDa band of bovine serumalbumin (BSA), which is a constituent of the 7H9 medium used for growingM. bovis, as well as several bands of greater than 45 kDa molecularmass, but not the bands characteristic of cytochrome c₅₅₁ or azurin.(See Example 9.)

Azurin and/or cytochrome C₅₅₁ and the QSFT fractions exhibitapoptosis-triggering cytotoxicity towards phagocytic cells. Inisolation, cytochrome C₅₅₁ causes cellular growth arrest. A purifiedazurin/cytochrome c₅₅₁ mixture, or the B. cepacia QSFT fraction, treatedwith a mixture of anti-azurin and anti-cytochrome C₅₅₁ antibodies, showgreatly diminished macrophage cytotoxicity. In contrast, the M. bovisQSFT fraction, when pretreated with anti-azurin/anti-cytochrome C₅₅₁antibodies, shows very little reduction in cytotoxicity, confirming thatM. bovis QSFT fraction contains cytotoxic factors other than azurin orcytochrome c₅₅₁. Thus different pathogens secrete differentapoptosis-inducing or cellular growth arresting cytotoxic factors, allof which would be excellent targets for drug development.

ATP-Independent Cytotoxic Factors

I. Cupredoxin Compounds

These small blue copper proteins (cupredoxins) are electron transferproteins (10-20 kDa) that participate in bacterial redox chains,photosynthesis or are of unknown function. The copper ion is solelybound by the protein matrix. A special distorted trigonal planararrangement to two histidine and one cysteinate ligands around thecopper gives rise to very peculiar electronic properties of the metalsite and an intense blue color. A number of cupredoxins have beencrystallographically characterized at medium to high resolution.

Azurin

The azurins are copper containing proteins of 128 amino acid residueswhich belong to the family of cupredoxins involved in electron transferin plants and certain bacteria. The azurins include those from P.aeruginosa (PA) (SEQ ID NO: 1), A. xylosoxidans, and A. denitrificans.Murphy, L. M. et al., J. Mol. Biol., vol. 315, pp 859-71 (2002), thecontents of which are incorporated for all purposes by this reference.Although the sequence homology between the azurins varies between60-90%, the structural homology between these molecules is high. Allazurins have a characteristic β-sandwich with Greek key motif and thesingle copper atom is always placed at the same region of the protein.In addition, azurins possess an essentially neutral hydrophobic patchsurrounding the copper site (Murphy et al.).

Plastocyanins

The plastocyanins are soluble proteins of eukaryotic plants that containone molecule of copper per molecule and are blue in their oxidized form.They occur in the chloroplast, where they function as electron carriers.Since the determination of the structure of poplar plastocyanin in 1978,the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) andplant (French bean) plastocyanins has been determined either bycrystallographic or NMR methods, and the poplar structure has beenrefined to 1.33 Å resolution. SEQ ID NO: 2 shows the amino acid sequenceof plastocyanin from Phormidium laminosum.

Despite the sequence divergence among plastocyanins of algae andvascular plants (e.g., 62% sequence identity between the Chlamydomonasand poplar proteins), the three-dimensional structures are conserved(e.g., 0.76 Å rms deviation in the C alpha positions between theChlamydomonas and Poplar proteins). Structural features include adistorted tetrahedral copper binding site at one end of aneight-stranded antiparallel beta-barrel, a pronounced negative patch,and a flat hydrophobic surface. The copper site is optimized for itselectron transfer function, and the negative and hydrophobic patches areproposed to be involved in recognition of physiological reactionpartners. Chemical modification, cross-linking, and site-directedmutagenesis experiments have confirmed the importance of the negativeand hydrophobic patches in binding interactions with cytochrome f, andvalidated the model of two functionally significant electron transferpaths in plastocyanin. One putative electron transfer path is relativelyshort (approximately 4 Å) and involves the solvent-exposed copper ligandHis-87 in the hydrophobic patch, while the other is more lengthy(approximately 12-15 Å) and involves the nearly conserved residue Tyr-83in the negative patch, Redinbo et al., J. Bioenerg. Biomembr., vol.26(1), pp 49-66 (1994) the contents of which are incorporated for allpurposes by this reference.

Rusticyanins

Rusticyanins are blue-copper containing single-chain polypeptidesobtained from a thiobacillus. The X-ray crystal structure of theoxidized form of the extremely stable and highly oxidizing cupredoxinrusticyanin from Thiobacillus ferrooxidans (SEQ ID NO: 3) has beendetermined by multiwavelength anomalous diffraction and refined to 1.9 Åresolution. The rusticyanins are composed of a core beta-sandwich foldcomposed of a six- and a seven-stranded b-sheet. Like other cupredoxins,the copper ion is coordinated by a cluster of four conserved residues(His 85, Cys138, His143, Met148) arranged in a distorted tetrahedron.Walter, R. L. et al., J. Mol. Biol., vol. 263, pp-730-51 (1996) thecontents of which are incorporated for all purposes by this reference.

Pseudoazurins

The pseudoazurins are a family of blue-copper containing single-chainpolypeptide. The amino acid sequence of pseudoazurin obtained fromAchromobacter cycloclastes is shown in SEQ ID NO: 4. The X-ray structureanalysis of pseudoazurin shows that it has a similar structure to theazurins although there is low sequence homology between these proteins.Two main differences exist between the overall structure of thepseudoazurins and azurins. There is a carboxy terminus extension in thepseudoazurins, relative to the aruzins, consisting of two alpha-helices.In the mid-peptide region azurins contain an extended loop, shortened inthe pseudoazurins, which forms a flap containing a short α-helix. Theonly major differences at the copper atom site are the conformation ofthe MET side-chain and the Met-S copper bond length, which issignificantly shorter in pseudoazurin than in azurin.

II Cytochrome C₅₅₁

Cytochrome C₅₅₁ from P. aeruginosa (Pa-C₅₅₁) is a monomeric redoxprotein of 82 amino-acid residues (SEQ ID NO: 5), involved indissimilative denitrification as the physiological electron donor ofnitrite reductase. The functional properties of Pa-C₅₅₁ have beenextensively investigated. The reactions with non-physiological smallinorganic redox reactants and with other macromolecules, like bluecopper proteins, eukaryotic cytochrome c and the physiological partnernitrite reductase have provided a test for protein-protein electrontransfer.

The three-dimensional structure of Pa-C₅₅₁, which is a member ofbacterial class I cytochromes, shows a single low-spin heme with His-Metligation and the typical polypeptide fold which however leaves the edgesof pyrrole rings II and III of the heme exposed (Cutruzzola et al., J.Inorgan. Chem., vol 88, pp 353-61 (2002) the contents of which areincorporated for all purposes by this reference). The lack of a20-residue omega loop, present in the manunalian class I cytochromes,causes further exposure of the heme edge at the level of propionate 13.The distribution of charged residues on the surface of Pa-C₅₅₁ is veryanisotropic: one side is richer in acidic residues whereas the otherdisplays a ring of positive side chains, mainly lysines, located at theborder of a hydrophobic patch which surrounds the heme crevice. Thispatch comprises residues Gly11, Val13, Ala14, Met22, Val23, Pro58,Ile59, Pro60, Pro62, Pro63 and Ala65. The anisotropic chargedistribution leads to a large dipolar moment which is important forelectron transfer complex formation.

The charge distribution described above for Pa-C₅₅₁ has been reportedfor other electron transfer proteins and their electron acceptors.Moreover, modification by site-directed mutagenesis of residues withinthe hydrophobic or charged patch has shown for different proteins theimportance of surface complementarity for binding and electron transfer.As an example, evidence for the relevance of the hydrophobic patch forthe electron transfer properties of azurin from P. aeruginosa came fromthe studies carried out on mutants of residues Met44 and Met64 changedto positively and negatively charged amino acids. (Cutruzzola et al.)

Induction of Apoptosis or Growth Arrest in Cancer Cells byATP-Independent Cytotoxic Factors

The present invention provides methods of using ATP-independentcytotoxic factors to induce apoptotic cell death or cellular growtharrest in cancer cells. ATP-independent cytotoxic factors, such as thecupredoxin compounds and cytochrome C₅₅₁, can be used to treatconditions related to an abnormal failure of cell death. It is wellknown that cancer cells are not prone to undergoing apoptosis. Inaccordance with one aspect of the present invention, administering acytotoxic factor or active agent stimulating cytotoxic factor secretionin an amount sufficient to induce cancer cell apoptosis or cellulargrowth arrest would be beneficial in reducing tumor size in vivo andretarding the growth of tumors. For example, tests comparing azurin andcytochrome C₅₅₁ to a known anti-melanoma cancer drug[5-(3,3′-N,N′-dimethyl triazen-1-yl)-imidazole-4-carboxyamide] (DTIC)show that a mixture of azurin and cytochrome C₅₅₁ provides a potent,non-toxic composition that promotes tumor regression in vivo in nudemice.

In one embodiment of the invention, a method is provided whereintreatment with a cupredoxin compound, such as azurin, induces apoptoticcell death in cancer cells. While not wishing to be bound by theory, itis believed that the cytotoxic activity of the cupredoxin compoundresults from its ability to form a complex with, and stabilize, thetumor suppressor protein p53. p53 acts as a “tumor suppressor” gene andits under production or inactivation through mutation can lead to tumordevelopment.

The half-life of p53 within a cell is normally only a few minutes.Stabilization of p53 allows the significant generation of reactiveoxygen species (ROS) which is a potent inducer of apoptosis. Azurinforms a complex with p53, stabilizes it, and enhances its intracellularlevel, thereby inducing apoptosis via caspase-3 and capase-9-dependentmitrochondrial pathways Yamada, T. et al., Infec. Immun., vol. 70, pp7054-62 (2002), the contents of which are incorporated for all purposesby this reference.

The redox activity of azurin is not critical for its cytotoxic activity.Instead, generation of reactive oxygen species during complex formationis the inducing factor for apoptosis. Goto, M. et al., Mol. Microbiol.,47, pp 549-59 (2003) the contents of which are incorporated for allpurposes by this reference. For example, apo-azurin, which has an aminoacid sequence SEQ ID NO: 1 but does not contain a copper atom, has amuch lower redox activity compared to azurin but demonstratessignificant cytotoxic activity.

The importance of complex formation with p53 is illustrated bydifferences in the cytotoxic activity of two mutant azurins, C112D (SEQID NO. 6) and the double mutant M44KM64E (SEQ ID NO. 7). The binding ofcopper to the Cys-112 residue is important for the redox activity. TheC112D mutant, which is defective in co-ordinating with copper, has aredox activity of approximately 0.01% of azurin but shows significantcytotoxicity. In comparison, the M44KM64E mutant has a redox activity ofapproximately 2% of azurin but shows little cytotoxicity.

The azurin molecule contains a hydrophobic patch that is the interactionsite of the physiological partners cytochrome C₅₅₁ and nitritereductase. (Cutruzzola et al.) The C112D mutant, in with the hydrophobicpatch is unchanged, is capable of forming complexes with p53 and raisingits intracellular level. However, the M44KM64E double mutant, where anelectric dipole is created in the hydrophobic patch, is not capable offorming such stable complexes. Thus, the interaction site withcytochrome C₅₅₁ and nitrite reductase is also important for complexformation with p53.

The glycerol gradient centrifugation and Glutathione S-transferase (GST)pull-down methods have been used to show the interaction of cupredoxincompounds with p53. Yamada et al. (2002), the contents of which areincorporated for all purposes by this reference. p53 is known to formoligomeric complexes and a GST-p53 fusion protein sediments at variousglycerol fractions, such as 5, 10, 15, 20, or 25% glycerol, while azurinsediments at 5% glycerol. Prior incubation of azurin with the GST-p53fusion protein followed by centrifugation in the glycerol gradientdemonstrates the presence of azurin in all glycerol fractions,indicating its association with p53. The C112D mutant, but not theM44KM64E mutant, showed similar association. Yamada et al. (2002).

Preincubation of the GST-p53 fusion protein with the M44KM64E mutantazurin altered p53 oligomerization, resulting in most of the GST-p53being found at 5 to 10% glycerol, where the mutant azurin protein wasalso found. This indicates that the hydrophobic patch of azurin is alsoinvolved in p53 interaction. A loss of azurin hydrophobicity not onlyresults in a loss in cytotoxicity but also interferes witholigomerization. Although the M44KM64E mutant shows little induction ofapoptosis, it does show significant inhibition of cell cycleprogression. Thus, a change in the nature of the p53-cupredoxin complexcan shift the specificity of p53 from apoptosis to cellular growtharrest.

The action of azurin is dependent upon the tumor cell having afunctional p53 tumor suppressor gene. However, cytotoxic factors canalso cause retardation in the growth of cells having a deficient p53tumor suppressor gene. For example, cytochrome C₅₅₁ does not act on p53but does significantly enhance the level of the tumor suppressor proteinp16. C₅₅₁ inhibits cell cycle progression on macrophages and alsoenhances the effect of azurin. In addition, combinations of cytotoxicfactors such as azurin and C₅₅₁ (or M44KM64E) can achieve more effectiveinhibition of tumor progression by inducing both apoptosis and growtharrest.

Because the mode of action of cytochrome C₅₅₁ is independent of thestatus of p53 in the cell, it provides for a method of cancer regressionin the 50% of human cancers that have a deficient in p53 tumorsuppressor gene. In addition to C₅₅₁, other cytochromes, for example,cytochrome f from cyanobacteria also demonstrate cytotoxicity.

Cytotoxic Factors in the Treatment of Infectious Disease

In another aspect of the present invention, characterization ofcytotoxic factors can be useful for identifying new substances thatinhibit cell death, for example, in an infectious disease. For example,inhibition of the secretion or activity of an ATP-utilizing cytotoxicfactor, or the production of ATP, can reduce or eliminate cytotoxicactivity by a disease-causing pathogen.

Accordingly, appropriately administering a compound that inhibits thesecretion or activity of a cytotoxic factor provides a useful tool foranti-infective development. Examples of active agents useful forinhibiting activity of cell death inducing cytotoxic factor can includeantibodies for cytotoxic factors, as well as analogues of ATP thatprevent the activation of ATP-utilizing enzymes. Examples of cytotoxicfactors and active agents for inhibiting or stimulating cytotoxic factorsecretion or expression include, but are not limited to, ATP-utilizingenzymes, redox proteins, activators of ATP-production, inhibitors of ATPproduction, activators of redox proteins, and inhibitors of redoxproteins.

Pharmaceutical Compositions Comprising Cytotoxic Factors

Pharmaceutical compositions comprising cytotoxic factors can bemanufactured in any conventional manner, e.g. by conventional mixing,dissolving, granulating, dragee-making, emulsifying, encapsulating,entrapping, or lyophilizing processes. The substantially pure cytotoxicfactor or other agent can be readily combined with a pharmaceuticallyacceptable carrier well-known in the art. Such carriers enable thepreparation to be formulated as a tablet, pill, dragee, capsule, liquid,gel, syrup, slurry, suspension, and the like. Suitable excipients canalso include, for example, fillers and cellulose preparations. Otherexcipients can include, for example, flavoring agents, coloring agents,detackifiers, thickeners, and other acceptable additives, adjuvants, orbinders.

The compositions of the invention can be used in treatment of acondition related to cell death or in the prevention thereof. Thesubstantially pure cytotoxic factor can be administered in an amountsufficient to prevent or treat a condition related to cell death.Typically, the host organism is a mammal, such as a human or animal.

Administration of Compositions Comprising Cytotoxic Factors

The compositions of the present invention can be administered by anysuitable route, for example, by oral, buccal, inhalation, sublingual,rectal, vaginal, transurethral, nasal, topical, percutaneous, i.e.,transdermal or parenteral (including intravenous, intramuscular,subcutaneous and intracoronary) administration. The compositions andpharmaceutical formulations thereof can be administered in any amounteffective to achieve its intended purpose. More specifically, thecomposition is administered in a therapeutically effective amount.

In various embodiments, the cytotoxic factor composition includescarriers and excipients (including but not limited to buffers,carbohydrates, mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents, suspendingagents, thickening agents and/or preservatives), water, oils, salinesolutions, aqueous dextrose and glycerol solutions, otherpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents, wetting agents and the like. It will be recognizedthat, while any suitable carrier known to those of ordinary skill in theart may be employed to administer the compositions of this invention,the type of carrier will vary depending on the mode of administration.Compounds may also be encapsulated within liposomes using well-knowntechnology. Biodegradable microspheres may also be employed as carriersfor the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252.

The compositions of the invention may be sterilized by conventional,well-known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration.

The cytotoxic factor compositions of the invention may be administeredin a variety of ways, including by injection (e.g., intradermal,subcutaneous, intramuscular, intraperitoneal and the like), byinhalation, by topical administration, by suppository, by using atransdermal patch or by mouth.

When administration is by injection, the cytotoxic factor may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks solution, Ringer's solution, orphysiological saline buffer. The solution may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Alternatively,the cytotoxic factor composition may be in powder form for constitutionwith a suitable vehicle, e.g., sterile pyrogen-free water, before use.

When administration is by inhalation, the cytotoxic factors may bedelivered in the form of an aerosol spray from pressurized packs or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or othersuitable gas. In the case of a pressurized aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount. Capsulesand cartridges of, e.g., gelatin for use in an inhaler or insufflatormay be formulated containing a powder mix of the proteins and a suitablepowder base such as lactose or starch.

When administration is by topical administration, the cytotoxic factorcomposition may be formulated as solutions, gels, ointments, creams,suspensions, and the like, as are well known in the art. In someembodiments, administration is by means of a transdermal patch. Whenadministration is by suppository (e.g., rectal or vaginal), cytotoxicfactor compositions may also be formulated in compositions containingconventional suppository bases.

When administration is oral, a cytotoxic factor composition can bereadily formulated by combining the cytotoxic factor withpharmaceutically acceptable carriers well known in the art. A solidcarrier, such as mannitol, lactose, magnesium stearate, and the like maybe employed; such carriers enable the chemotaxin to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, cellulosepreparation, granulating agents, and binding agents.

Nucleic acid molecules encoding cytotoxic factors, can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (Nabel et al., U.S. Pat. No. 5,328,470 1994. USA), or bystereotactic injection (Chen et al., Proc Natl Acad Sci USA, vol. 91, pp3054-7 (1994). The pharmaceutical preparation of a gene therapy vectorcan include an acceptable diluent or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g., retroviral vectors, the pharmaceutical preparation caninclude one or more cells that produce the gene delivery system.

Other convenient carriers, as well-known in the art, also includemultivalent carriers, such as bacterial capsular polysaccharide, adextran or a genetically engineered vector. In addition,sustained-release formulations that include cytotoxic factor moleculesallow for the release of cytotoxic factors over extended periods oftime, such that without the sustained release formulation, the cytotoxicfactor would be cleared from a subject's system, and/or degraded by, forexample, proteases and simple hydrolysis before eliciting or enhancingan therapeutic effect.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active cytotoxic factor which are sufficient to maintaintherapeutic effect. Generally, the desired cytotoxic factor isadministered in an admixture with a pharmaceutical carrier selected withregard to the intended route of administration and standardpharmaceutical practice. Pharmaceutical compositions used in accordancewith the present invention can be formulated in a conventional mannerusing one or more physiologically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the cytotoxicfactor, active agents, for inhibiting or stimulating the secretion ofcytotoxic factors, or a mixture thereof into preparations which can beused therapeutically.

In one aspect, the cytotoxic factor is delivered as DNA such that thepolypeptide is generated in situ. In one embodiment, the DNA is “naked,”as described, for example, in Ulmer et al., Science, vol. 259,pp-1745-49 (1993) and reviewed by Cohen, Science, vol. 259, pp-1691-92(1993). The uptake of naked DNA may be increased by coating the DNA ontoa carrier, e.g. biodegradable beads, which is efficiently transportedinto the cells. In such methods, the DNA may be present within any of avariety of delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacterial and viralexpression systems. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.See, e.g., WO90/11092, WO93/24640, WO 93/17706, and U.S. Pat. No.5,736,524.

Vectors, used to shuttle genetic material from organism to organism, canbe divided into two general classes: Cloning vectors are replicatingplasmid or phage with regions that are non-essential for propagation inan appropriate host cell and into which foreign DNA can be inserted; theforeign DNA is replicated and propagated as if it were a component ofthe vector. An expression vector (such as a plasmid, yeast, or animalvirus genome) is used to introduce foreign genetic material into a hostcell or tissue in order to transcribe and translate the foreign DNA,such as the DNA of a cytotoxic factor. In expression vectors, theintroduced DNA is operably-linked to elements such as promoters thatsignal to the host cell to transcribe the inserted DNA. Some promotersare exceptionally useful, such as inducible promoters that control genetranscription in response to specific factors. Operably-linking acytotoxic factor polynucleotide to an inducible promoter can control theexpression of the cytotoxic factor polypeptide or fragments. Examples ofclassic inducible promoters include those that are responsive toα-interferon, heat shock, heavy metal ions, and steroids such asglucocorticoids (Kaufman, Methods Enzymol., vol. 185, pp. 487-511(1990)) and tetracycline. Other desirable inducible promoters includethose that are not endogenous to the cells in which the construct isbeing introduced, but, however, are responsive in those cells when theinduction agent is exogenously supplied. In general, useful expressionvectors are often plasmids. However, other forms of expression vectors,such as viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses) are contemplated.

Vector choice is dictated by the organism or cells being used and thedesired fate of the vector. In general, vectors comprise signalsequences, origins of replication, marker genes, enhancer elements,promoters, and transcription termination sequences.

Kits Comprising Cytotoxic Factors

In one aspect, the invention provides kits containing one or more of thefollowing in a package or container: (1) a biologically activecomposition comprising a cytotoxic factor; (2) a pharmaceuticallyacceptable adjuvant or excipient; (3) a vehicle for administration, suchas a syringe; (4) instructions for administration. Embodiments in whichtwo or more of components (1)-(4) are found in the same container arealso contemplated.

When a kit is supplied, the different components of the composition maybe packaged in separate containers and admixed immediately before use.Such packaging of the components separately may permit long-term storagewithout losing the active components' functions.

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized cytotoxicpolypeptide or polynucleotide, or buffers that have been packaged undera neutral, non-reacting gas, such as nitrogen. Ampules may consist ofany suitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, etc., ceramic, metal or any other materialtypically employed to hold similar reagents. Other examples of suitablecontainers include simple bottles that may be fabricated from similarsubstances as ampules, and envelopes, that may comprise foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, or the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to be mixed. Removable membranes maybe glass, plastic, rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audiotape, etc. Detailed instructions may not bephysically associated with the kit; instead, a user may be directed toan internet web site specified by the manufacturer or distributor of thekit, or supplied as electronic mail.

Stimulation and Inhibition of the Secretion of Cytotoxic Factors.

The identification and characterization of the cytotoxic factors alsocan lead to the development of methods of stimulating of cytotoxicfactor secretion. Pathogenic organisms have been shown to secrete largeamounts of cytotoxic factors in the presence of mammalian proteins. Thisprinciple can be modified in the human body to provide new methods ofstimulating desired, or inhibiting undesired, cytotoxic factorproduction. Such methods are useful for inhibiting or stimulating cellapoptosis or causing cellular growth arrest. An understanding ofcytotoxic factors, and the characterization and development thereof,also allows for drug development and screening of active agents orcompounds suitable for modulating the cytotoxic factor activity orsecretion. The understanding of the secretion machinery related tocytotoxic factor secretion in cells additionally provides new avenues ofdeveloping and identifying the design of useful inhibitors orstimulators of cytotoxic factors. The delineation and identification ofthe presence of receptors for mammalian proteins also can be used as ameans to differentiate between the virulent and avirulentmicroorganisms, which can provide specificity in treating the disease.

Modification of Cytotoxic Factors.

Cytotoxic factors also can be chemically modified or genetically alteredto produce variants that lack an ATP-utilizing enzyme or redox activity,but retain toxicity. Mutations and/or truncations of cytotoxic factorscan produce cytotoxic agents of varying compositions also demonstratingfunctional activity. In particular, truncated derivatives with highefficacy and low antigenicity can be produced from the originalcytotoxic factor. Such modified or altered cytotoxic factors also areincluded in the scope of the present invention.

Various derivatives of cytotoxic factors may be synthesized by standardtechniques. Derivatives are amino acid sequences formed from nativecompounds either directly or by modification or partial substitution.Analogs are amino acid sequences that have a structure similar, but notidentical, to the native compound but differ from it in respect tocertain components or side chains. Analogs may be synthesized or from adifferent evolutionary origin.

Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified amino acid. Derivatives oranalogs of the cytotoxic factors include, but are not limited to,molecules comprising regions that are substantially homologous to thecytotoxic factors by at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%,or 99% identity over an amino acid sequence of identical size or whencompared to an aligned sequence in which the alignment is performed by ahomology algorithm.

In addition to naturally-occurring allelic variants of cytotoxicfactors, changes can be introduced by mutation into cytotoxic factorsthat incur alterations in the amino acid sequences of the encodedcytotoxic factors that do not significantly alter the cytotoxicactivity. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequences of the cytotoxic factors withoutaltering biological activity, whereas an “essential” amino acid residueis required for such biological activity. For example, amino acidresidues that are conserved among the cytotoxic factors of the inventionare predicted to be particularly non-amenable to alteration. Amino acidsfor which conservative substitutions can be made are well known in theart.

Useful conservative substitutions are shown in Table 1, “Preferredsubstitutions.” Conservative substitutions whereby an amino acid of oneclass is replaced with another amino acid of the same type fall withinthe scope of the invention so long as the substitution does notmaterially alter the biological activity of the compound.

TABLE 1 Preferred substitutions Preferred Original residue Exemplarysubstitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln,Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L)Norleucine, Ile, Val, Met, Ala, Ile Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu Norleucine

Non-conservative substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge, (3) hydrophobicity, or (4) the bulk of the side chain of thetarget site can modify the cytotoxic factor function. Residues aredivided into groups based on common side-chain properties as denoted inTable 2. Non-conservative substitutions entail exchanging a member ofone of these classes for another class. Substitutions may be introducedinto conservative substitution sites or more preferably intonon-conserved sites.

TABLE 2 Amino acid classes Class Amino acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain Gly, Pro conformationaromatic Trp, Tyr, Phe

The variant polypeptides can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis (Carter,Biochem J., vol. 237, pp 1-7 (1986); Zoller and Smith, Methods Enzymol.,vol. 154, pp 329-50 (1987)), cassette mutagenesis, restriction selectionmutagenesis (Wells et al., Gene, vol. 34, pp 315-23 (1985)) or otherknown techniques can be performed on the cloned DNA to produce thecytotoxic factor variant DNA.

The cytotoxic activity of the C112D and M44KM64E cytotoxic factormutants is described above. In addition, Example 19 shows the cytotoxicactivity of a number of chimeric azurin mutants prepared bysite-directed mutagenesis as described in Example 18. The presentinvention can also utilize cytotoxic factors, such as apo-azurin, inwhich a copper atom is not present. Both apo-azurin and the C112D mutantshow significant cytotoxic activity whereas the M44KM64E mutant doesnot. However, the M44KM64E mutant does cause significant inhibition ofcell cycle progression.

One embodiment of the present invention utilizes mutated cytotoxicfactors retaining the ability to form a complex with and stabilize p53and hence induce apoptosis. In another embodiment, the present inventionutilizes mutated cytotoxic factors, such as the M44KM64E mutant, havingthe ability to interact with p53 and cause cellular growth arrest.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof, and,therefore, only such limitations should be imposed as are indicated bythe appended claims.

EXAMPLES Example 1 Stimulation of the Secretion of Cytotoxic Factors byMammalian Proteins

Clinical and environmental isolates (five of each) of B. cepacia weregrown in proteose peptone-yeast extract (PPY) broth with and withoutadded α2-macroglobulin (1 mg/ml). After growth for 10 hours at 34° C. ona shaker, a portion of the growth medium from each culture wascentrifuged and the supernatant filtered through a 0.22 μm milliporefilter to remove whole cells and debri. The filtered supernatant wasthen tested for adenylate kinase activity as described in Melnikov A. etal., Mol. Microbiol., 36: 1481-1493 (2000). Adenylate kinase transfersthe terminal phosphate from [γ-³²P]ATP to AMP giving rise to ADP. Theproducts of this reaction were then detected by thin-layerchromatography. Secretion of adenylate kinase was minimal when B.cepacia cells were grown in PPY broth. However, secretion from theclinical isolates, but not for the environmental isolates, wasstimulated in the presence of α2-macroglobulin.

Immunofluorescence microscopy with anti-α2-macroglobulin antibody showedthat the clinical isolates had receptors that bound α2-macroglobulinwhile the environmental isolates lacked such receptors. The clinical andenvironmental isolates of B. cepacia were grown in absence or inpresence of 1 mg/ml α2-macroglobulin in PPY broth for 1 hr. Extraneousα2-macroglobulin was removed by washing with phosphate-buffered saline.The cells were incubated for 2 hours with fluorescein isothiocyanate(FITC)-conjugated α2-macroglobulin antibodies, obtained by injectingrabbits with α2-macroglobulin. After washing with phosphate-bufferedsaline, the FITC conjugated antibody treated cells were fixed in 16%paraformaldehyde, coated on poly-L-lysine coated slides, and examined byconfocal microscopy. Only the clinical isolates that showed enhancedcytotoxic factor secretion in the presence of α2-macroglobulinfluoresced (green fluorescing cells), demonstrating the presence of thereceptors for α2-macroglobulin.

Example 2 ATP-dependent Macrophage Killing by Filtered Supernatant orColumn Chromatographic Fractions Derived from B. cepacia Growth Medium

A clinical strain of B. cepacia (strain 38—collection number 95828, D.G. Allison, University of Manchester Institute of Science andTechnology, Manchester, UK) was grown in TB broth (10 g of Bactotryptone, 3 g of Bacto beef extract per liter of water) at 34° C. on ashaker to an OD_(550 nm) of 1.3. The growth medium was then centrifugedand the supernatant filtered through a 0.22 μm millipore filter toremove whole cells and debri. Macrophage cells were isolated from J774cell lines and grown in RPMI medium 1640 (GIBRO-BRL, Grand Island, N.Y.)as described by Zaborina O. et al., Infect. Immun. 67: 5231-5242 (1999).The filtered growth medium was added to hydroxyapatite, ATP-agarose, andQ-sepharose columns in sequence. The flow-through fraction from thehydroxyapatite column (HAFT) was fractionated on the ATP-agarose column(AAFT). The AAFT fraction was then fractionated on the Q-sepharosecolumn (QSFT).

10⁶ macrophages were added to wells in a 96 well plate and incubated fortwo hours in a CO₂ incubator for attachment. 2 μg of protein from thesupernatant or the flow-through fraction from each of the above columnswas added to the wells and the plates incubated for 4 hrs in thepresence or absence of 1.0 mM ATP. The extent of macrophage cell deathwas then measured by the release of the intracellular enzyme lactatedehydrogenase (LDH) as described by Zaborina O. et al., Infect. Immun.,67: 5231-5242 (1999). The extent of macrophage killing, in the presenceand in the absence of 1.0 mM ATP, by the filtered supernatant (SUP) andthe HAFT, AAFT and QSFT column fractions is shown in FIG. 1. All assayswere carried out in triplicate and error bars are indicated.

Example 3 ATP-independent Macrophage Killing by Filtered Supernatant orColumn Chromatographic Fractions Derived from B. cepacia Growth Medium

The supernatant (SUP) and column chromatographic fractions (HAFT, AAFTand QSFT) of B. cepacia growth medium were as in Example 2. Macrophageisolation was as in Example 2. The extent of macrophage cell death hasbeen determined by release of LDH as in Example 2 and is shown in FIG.2. Only the QSFT fraction shows high ATP-independent cytotoxicitytowards macrophages.

Example 4 Induction of Apoptosis in Macrophages by P. aeruginosaCytotoxic Factor

P. aeruginosa was grown in L broth at 37° C. for 12 hours to anOD_(550 nm) of 1.2. The growth medium was then centrifuged and thesupernatant filtered through a 0.22 μm filter. Supernatant (SUP) andcolumn chromatographic fractions (HAFT, AAFT and QSFT) were collected asin Example 2. Macrophage isolation was as in Example 2. 2 μg of proteinfrom the supernatant or one of the flow-through fractions was added to1×10⁵ macrophages in 200 μl of RPMI medium and the mixture incubatedovernight. Induction of apoptosis in macrophages either untreated ortreated by overnight incubation with the SUP or the HAFT, AAFT or QSFTfractions was measured by confocal microscopy using the ApoAlertMitochondria Membrane Sensor kit (Clontech Laboratories, Inc., PaloAlto, Calif., U.S.A.) as described by Zaborina O. et al., Microbiology146: 2521-2530 (2000).

In this assay, healthy, non-apoptotic cells fluoresce red whileapoptotically dead cells fluoresce green. A combination of red and greenproduces yellow fluorescing cells, indicating apoptotically dying cells.Nontreated macrophages or macrophages treated overnight with the SUP,HAFT or AAFT fractions fluoresced primarily red, indicating a lack ofapoptotic cell death. Macrophages treated overnight with the QSFTfraction fluoresced mostly green, indicating the apoptotic death of mostof the macrophages. A time course study showed that apoptosis set in atabout 6 hours (indicated by a combination of red and green fluorescencemaking the cell yellow) and was complete in 12 to 16 hours.

Example 5 Induction of Apoptosis in Mast Cells by B. cepacia CytotoxicFactors

Mast cells were isolated by the method described by Melnikov A. et al.,Mol. Microbiol. 36: 1481-1493 (2000). B. cepacia fractionated growthmedium was prepared as in Example 2. Induction of apoptosis in mastcells by B. cepacia cytotoxic factor was determined using confocalmicroscopy, as described in Example 4.

Nontreated mast cells or mast cells, treated overnight with the SUP,HAFT or AAFT fractions of B. cepacia growth medium, fluoresced primarilyred, indicating a lack of apoptotic cell death. Mast cells treatedovernight with the QSFT fraction of B. cepacia growth medium fluorescedmostly green, indicating the apoptotic death of most of the mast cells.

Example 6 Induction of Apoptosis in Macrophages by B. cepacia and M.bovis QSFT Fractions

Macrophage isolation was as in Example 2. Induction of apoptosis inmacrophages by B. cepacia and M. bovis cytotoxic factors was determinedusing the methods of Example 4. Induction of apoptosis of macrophageswas observed when they were treated with the B. cepacia and M. bovisQSFT fractions.

Example 7 Measurement of Caspase Activities (caspase-3 and caspase-9) inthe Cytosolic Extracts of Macrophages Treated with the B. cepacia QSFTFraction

Macrophage isolation was as in Example 2. Macrophages are treatedovernight with the B. cepacia QSFT fraction using the method describedin Example 2. The preparation of macrophage cytoslic extract and thecaspase assays were as described by Zaborina O. et al., Microbiology146: 2521-2530 (2000).

Briefly, determination of caspase-3 activity was performed usingAc-DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-p-NO₂-aniline) as a substrate.Release of pNA (p-nitroaniline) was determined spectrophotometrically at405 nm from the caspase-3 substrate (200 μm) after 15, 30, 45, 60, 75and 90 min incubation at 37° C. (FIG. 3A) with uninduced macrophagecytosolic extract; cytosolic extract of macrophages incubated overnightwith the B. cepacia QSFT fraction (10 μg protein); and cytosolic extractof macrophages incubated overnight with the B. cepacia QSFT fraction (10μg protein) and added inhibitor (DEVD-CHO). 10 μg of macrophagecytosolic protein was used in each case.

In the caspase-9 assay, release of pNA from 200 μM of the caspase-9substrate Ac-LEHD-pNA (N-acetyl-Leu-Glu-His-Asp-p-NO₂-aniline) wasdetermined, after 15, 30, 45, 60, 75 and 90 min incubation (FIG. 3B),with uninduced macrophage cytosolic extract, cytosolic extract ofmacrophages incubated overnight with the B. cepacia QSFT fraction (10 μgprotein) and cytosolic extract of macrophages incubated overnight withthe B. cepacia QSFT fraction (10 μg protein) plus inhibitor (LEHD-CHO).10 μg of macrophage cytosolic protein was used in each case.

DEVD-CHO and LEHD-CHO respectively block Caspase 3 and Caspase 9activity and are available from Biomol Research Laboratories, PlymouthMeeting, Pa., U.S.A. The activities of both caspase-9 and caspase-3increased when macrophages were treated overnight with the B. cepaciaQSFT fraction (FIGS. 3A and B). These activities remained very low foruntreated macrophages or with inhibitor present, suggesting that theinduction of apoptosis by the QSFT fractions involves caspaseactivation.

Example 8 TUNEL Assay to Measure Nuclear DNA Fragmentation inMacrophages Treated with M. bovis or B. cepacia QSFT Fractions

Fractionated B. cepacia growth medium was obtained using the methoddescribed in Example 2. M. bovis BCG was grown in Middlebrook 7H9 broth(Difco Laboratories, Maryland, U.S.A.) supplemented with 2% glycerol,0.02% TWEEN® 80 and ADC (ablumin/dextrose/citrate) (available from DifcoLaboratories, Maryland, U.S.A.). The bacteria were grown for severaldays at 32° C. on a shaker before harvesting. Fractionated M. bovisgrowth medium was obtained using the method described in Example 2.Macrophage isolation was as in Example 2. Induction of apoptosis inmacrophages either untreated or treated by overnight incubation of theSUP or the HAFT, AAFT or QSFT fractions was measured using confocalmicroscopy by detecting apoptosis-induced nuclear DNA fragmentation withthe ApoAlert DNA fragmentation kit (Clontech Laboratories, Inc., PaloAlto, Calif., U.S.A.). This assay is based on terminaldeoxynuclotidyltransferase (Tdt)—mediated dUTP nick-end labeling(TUNEL), where Tdt catalyzes the incorporation of fluorescein-dUTP atthe free 3′-hydroxyl ends of fragmented DNA in cells undergoingapoptosis. The incorporation of fluorescein-dUTP in the fragmentednuclear DNA generates green fluorescence which is detected by confocalmicroscopy.

Macrophages treated with either the M. bovis or B. cepacia QSFTfractions showed a yellow-green nucleus in the red cytoplasmicbackground, indicating nuclear DNA fragmentation. Little or nofragmentation was observed with untreated macrophages or withmacrophages treated with other column fractions.

Example 9 SDS-PAGE Analysis of Proteins in the Supernatant and the AAFT,HAFT and QSFT Fractions of Growth Media from P. aeruginosa, B. cepaciaand M. bovis

SDS-PAGE separation showed the proteins present in the supernatant andthe AAFT, HAFT and QSFT Fractions of P. aeruginosa, B. cepacia and M.bovis. The QSFT medium fraction from mucoid P. aeruginosa strain 8821showed the presence of two bands, a 18 kDa band corresponding to azurinby N-terminal analysis and a 9 kDa band corresponding to cytochromec₅₅₁. The B. cepacia QSFT fraction showed the presence of threepredominant bands of 75 kDa, 20 kDa and 8 kDa. The N-terminal amino acidsequence of 10 amino acids of the 20 kDa band (AHHSVDIQGN), determinedby Edman degradation, showed 80% sequence homology to that of theN-terminal 10 amino acid sequence of P. aeruginosa azurin while theN-terminal amino acid sequence of 10 amino acids of the 8 kDa band(EDPEVLFKNK) showed 100% match with that of P. aeruginosa cytochromec₅₅₁. Thus the QSFT fractions having high cytotoxic activity of both P.aeruginosa and B. cepacia show enrichment with azurin and cytochromec₅₅₁ type of redox proteins. In contrast, the M. bovis QSFT fractionshowed a thick 65 kDa band of bovine serum albumin (BSA), which is aconstituent of the 7H9 medium used for growing M. bovis, as well asseveral bands of greater than 45 kDa molecular mass, but not the 8 kDaor 22 kDa cytochrome c₅₅₁ or azurin type of proteins.

Example 10 Cell Death in Macrophages Treated with Azurin/Cytochrome c₅₅₁

Purified azurin and cytochrome c₅₅₁ (Sigma Chemicals, St. Louis U.S.A.)were added to macrophages, prepared as in Example 2, and the mixtureincubated for 2 hrs. Azurin and cytochrome c₅₅₁ concentrations were asin FIG. 4. The numbers represent μg protein. Macrophage cell death wasmeasured by the release of the intracellular enzyme lactatedehydrogenase (LDH) using the method of Example 2. Both azurin andcytochrome c₅₅₁ caused macrophage cell death. A combination of azurinand cytochrome c₅₅₁ caused more extensive macrophage cell death. Thebuffer control (buffer) is shown at right. (FIG. 4).

Example 11 Induction of Apoptosis in Macrophages Treated withAzurin/Cytochrome c₅₅₁

Macrophage isolation was as in Example 2. The macrophages were treatedwith azurin/cytochrome C₅₅₁ (50/25 μg) for 4 and 6 hours and thenexamined by confocal microscopy, using the ApoAlert MitochondriaMembrane Sensor kit as in Example 4, to determine the extent ofapoptosis. Macrophages underwent increasing levels of apoptosis withincreasing periods of incubation in presence of azurin/cytochrome c₅₅₁mixture. Control macrophages without treatment (treated withphosphate-buffered saline for 6 hours) did not show apoptosis.

Example 12 Cytotoxicity of an Azurin/Cytochrome C₅₅₁ Mixture or the QSFTFractions Derived from B. cepacia or M. bovis in Macrophages afterPretreatment with Anti-azurin and Anti-cytochrome c₅₅₁ Antibodies

Macrophage isolation was as in Example 2. Macrophages were treated witha purified azurin/cytochrome c₅₅₁ mixture (50/25 μg), or the B. cepaciaor M. bovis QSFT fractions in the presence and absence of a mixture ofanti-azurin and anti-cytochrome c₅₅₁ antibodies prepared in rabbits.Antibodies were mixed in a ratio of 1:1 and the mixed antibody (1, 2, 3,or 4 mg) was used for treatment of macrophages.

The extent of macrophage cell death was determined by release of the LDHas in Example 2. FIG. 5 shows a reduction of cytotoxicity towardsmacrophages treated with an azurin/cytochrome c₅₅₁ mixture (A+C), or theQSFT fraction derived from B. cepacia (Bc-QSFT), when anti-azurin andanti-cytochrome c₅₅₁ antibodies are present. This reduction was notobserved with the QSFT fraction from M. bovis (Mb-QSFT).

Hence, when an azurin/cytochrome c₅₅₁ mixture or the B. cepacia QSFTfraction was treated with a mixture of anti-azurin and anti-cytochromec₅₅₁ antibodies, and then assayed for macrophage cytotoxicity, thecytotoxicity was greatly diminished. In contrast, when the M. bovis QSFTfraction, which was previously shown by SDS-PAGE gel to lack azurin andcytochrome c₅₅₁ bands (Example 9), was pretreated withanti-azurin/anti-cytochrome c₅₅₁ antibodies and then assayed forcytotoxicity, very little reduction in cytotoxicity was observed.

Example 13 Induction of Apoptosis in Tumor Cell Lines by the B. cepaciaQSFT Fraction and by Azurin/Cytochrome c₅₅₁ as Measured by ConfocalMicroscopy

H460 lung carcinoma, PA-1 ovarian cancer, NCF breast cancer, HT-29 coloncancer and HT-1080 leukemia cell lines were obtained from the AmericanType Culture Collection (Manassas, Va., U.S.A.). MDD7 and MN1 breastcancer cell lines were obtained from Andrei Gudkov, Ph.D., ClevelandClinic Foundation (Cleveland, Ohio U.S.A.). UISO-BCA-9 breast cancer andUISO-MEL-1, MEL-2, MEL-6 and MEL-29 melanoma cell lines were developedand maintained as described in Rauth, S et al., In vitro Cellular andDevelopmental Biology, 30a(2): 79-84 (1994) and Rauth, S et al.,Anticancer Research, 14(6): 2457-2463 (1994). Approximately 1×10⁵ ofeach cell type were cultured overnight in a 0.15 mm thick dTC3 dish(Bioptech, Butler, Pa., U.S.A.) in the presence of the B. cepacia QSFTfraction (5 μg protein) or a azurin/cytochrome c₅₅₁ mixture (50/25 μg).The cells were subsequently examined by confocal microscopy, as inExample 4, to determine the extent of apoptosis. Both the B. cepaciaQSFT fraction the and azurin/cytochrome c₅₅₁ mixture induced extensiveapoptosis in H460 lung carcinoma, HT-29 colon cancer, HT-1080 leukemia,PA-1 ovarian cancer, MDD7, NCF and MN1 breast cancer, and USIO-MEL-1,MEL-2, MEL-6 and MEL-29 melanoma cells after overnight incubation. Ineach case, cells not treated with cytotoxic factor (phosphate-bufferedsaline added) did not show extensive apoptosis.

Example 14 Induction of Apoptosis in USIO-Mel-6 Melanoma Cell Line bythe M. bovis QSFT Fraction as Measured by TUNEL Assay

USIO-Mel-6 melanoma cells were prepared as described in Rauth, S et al.,Anticancer Research, 14(6): 2457-2463 (1994). M. bovis QSFT fraction wasprepared as in Example 8. The melanoma cells treated with M. bovis QSFTfraction (5 μg protein) and untreated control cells were incubated for12 hours. Induction of apoptosis was measured using the TUNEL assay todetect apoptosis-induced nuclear DNA fragmentation as in Example 8.Melanoma cells treated with the M. bovis QSFT fraction showed ayellow-green nucleus in the red cytoplasmic background, indicatingnuclear DNA fragmentation. Little or no fragmentation was observed withuntreated melanoma cells.

Example 15 Reduction of Growth of Melanoma Tumor Cells (USIO-Mel-2) inNude Mice after Treatment with Azurin/Cytochrome C₅₅₁

Approximately 10⁶ USIO-Mel-2 cells were injected subcutaneously in nudemice (available from Frederick Cancer Research and Development Center,Frederick, Md. U.S.A.). Small tumors developed after approximately threeweeks. The mice then received once weekly intraperitoneal injections ofa known anti-melanoma drug, DTIC [5-(3,3′-N,N-dimethyltriazen-1-yl)-imidazole-4-carboxamide] (7.5 μg) (see Ahlmais et al.,Cancer 63: 224-7 (1989)) or three weekly intraperitoneal injections of ahigh (150 μg azurin/75 μg cytochrome c₅₅₁), low (10 μg azurin/5 μgcytochrome C₅₅₁) dose of azurin/cytochrome C₅₅₁ mixture or control(citrate buffer) for 4 weeks. The tumor volume was determined atintervals in the control, DTIC-treated, and high and low doseazurin/cytochrome c₅₅₁-treated mice.

The increases in tumor size in control, DTIC-treated andazurin/cytochrome c₅₅₁-treated nude mice are shown in FIG. 6 and theweight gain/loss data in such mice are shown in FIG. 7. Post-injectionof a high dosage of 150 μg azurin/75 μg cytochrome c₅₅₁ produced delayedgrowth and a shrinkage of the tumor size comparable of DTIC. FIG. 7shows that the injection of either DTIC or azurin/cytochrome C₅₅₁mixture did not affect the weight gain of the mice. All mice gainedweight during the experimental period.

Example 16 Effect of Post Injection of Azurin and M. bovis QSFT fractionin Nude Mice on Tumor Size after Injection of Melanoma Tumor Cells(Mel-6)

Approximately 10⁶ USIO-Mel-6 cells were injected subcutaneously in 3nude mice (available from Frederick Cancer Research and DevelopmentCenter, Frederick, Md. U.S.A.). Small tumors developed afterapproximately three weeks. One mouse was then injected intraperitoneallywith phosphate-buffered saline (control), one mouse was injected with M.bovis QSFT fraction (5 μg protein) and one mouse was injected with amixture of M. bovis QSFT fraction (5 μg protein) and Azurin (50 μg). TheM. bovis QSFT fraction was prepared as in Example 8. The sizes (tumorvolume) of the tumors in control, M. bovis QSFT fraction treated and M.bovis QSFT fraction/Azurin treated mice were determined over a period of30 days. These data are shown in FIG. 8. Both the treated nice showeddecreased tumor growth compared to the control mouse.

Example 17 Azurin induces Apoptosis and Regression of Human BreastCancer Cells

The human breast cell lines MCF-7 (p53+/+) and MDA-MB-157 (p53−/−) wereobtained from the stock culture collection of the Department of SurgicalOncology, University of Illinois at Chicago (UIC), Chicago. Normalbreast cells (MCF-10F) and skin cells were from the same source. HBL100cells were a gift from Dr. Nita J. Mahile, Department of Biochemistryand Molecular Biology, Mayo Clinics, Rochester, Minn. The cells weregrown either in MEM medium supplemented with Earle's salt, 10% FBS,Penicillin/Streptomycin or Macoy's 5A medium. The cells were grown at37° C. in 6% CO₂.

The azurin-encoding gene of Pseudomonas aeruginosa was amplified andcloned in pUC19. Azurin was purified from E. coli μM109 as described inYamada, T. et al., Infect. Immun., vol. 70, pp 7054-62 (2002), thecontents of which are incorporated for all purposes by this reference.

Cytotoxicity of azurin towards cell lines was determined using the MTTassay as described in Yamada et al., (2002). FIG. 9 shows thecytotoxicity of azurin for MCF-7 and MDA-MB-157 cells treated withvarious concentrations of azurin for 72 hours. After 72 hours oftreatment, azurin at a concentration of 28.5 μM (400 μg/ml) induced 50%cell death in MCF-7 cells within 72 hours. Under the same experimentalconditions, MDA-MB-157 cells required 57 μM (800 μg/ml) for 50% celldeath.

To determine whether azurin induces similar cell death in normal cells,two mammary epithelial cell lines were tested (HBL 100 and MCF-10F).After 72 hours of incubation with 57 μM (800 μg/ml) of azurin, only 20%of MCF-10F cells and 18% of HBL100 cells were nonviable. Cell viabilitywas determined by a cell titer 96 aqueous proteolytic assay (Eilon, G.F. et al., Cancer Chemother. Pharmacol., vol. 45, pp 183-91 (2001) usinga kit from Promega (Madison, Wis.).

Example 18 Treatment with Azurin Reduces Tumor Size in Nude MiceInjected with Breast Cancer Tumor Cells

Approximately 500,000 MCF-7 cells, obtained as in Example 17, wereinjected in the right lowest mammary fat pad of estradiol-pretreatedfemale nude mice (available from Frederick Cancer Research andDevelopment Center, Frederick, Md. U.S.A.). The mice were randomized intwo groups of 10 mice each. The treated group received 1 mg of azurin in1 ml of normal saline intraperitoneally daily for 28 days, and thecontrol group received 1 ml of saline daily for 28 days.

The treatment started three days after MCF-7 inoculation. During thecourse of the experiment, the mice were examined daily, 3-axis tumorvolume and body weights were measured twice weekly. On the 29th day, theanimals were sacrificed and detailed necropsy was performed. All thetumors and viscera were preserved for histological andimmunocytochemical examination.

Tumor volume in the mice treated daily with 1 mg of azurin for 28 dayshad a substantially slower rate of increase than in the animals in thecontrol group. Univariate analysis of the data showed that thedifference in growth rates of the tumor in these two groups(azurin-treated versus control) is significant. For example, 22 daysafter the start of treatment, the mean tumor volume in treated mice wasonly 22% of the mean tumor volume for the control mice (i.e., 0.0267 cm³and 0.1240 cm³ respectively, P=0.0179, Kruskal-Wallis test),demonstrating a 78% tumor growth inhibition.

At the conclusion of the experiment on the 29^(th) day, the mean tumorvolume in the azurin-treated group was only 15% of the mean tumor volumeof the control group. This is further illustrated by FIG. 10 showing thegraph of the variation over time of mean tumor volumes for the twogroups, expressed in cm³.

In the multivariate approach, nonlinear mixed-effect models were fittedto the data. One model that was fitted for tumor growth was exponentialin time, with coefficients that were subject-specific mixed effects. Forthe control group, the fitted model was: tumor volume=exp{−4.23+0.06*time}, while for the treated group, it was: tumor volume=exp{−4.23+0.03*time}. The difference was statistically significant(P=0.0456). During the period of treatment (28 days), the treatedanimals did not show any sign of toxicity as evidenced by weight lossand/or other commonly observed signs of toxicity.

The extent of apoptosis in tumors was estimated by TUNEL stain as inExample 8. The azurin-treated group showed a marked increase inapoptotic figures as compared to the controls, where apoptotic cellswere rarely encountered.

Example 19 Preparation of Azurin Mutants

Microorganism and Plasmids

The azurin gene (wild type azurin) was amplified by polymerase chainreaction (PCR) according to the method described by Kukimoto et al.,FEBS Lett, vol. 394, pp 87-90 (1996), the contents of which areincorporated for all purposes by this reference. PCR was performed usinggenomic DNA from P. aeruginosa strain PAO1 as a template DNA. Theforward and reverse primers used were 5′-GCCCAAGCTTACCTAGGAGGCTGCTCCATGCTA-3′ (SEQ ID NO: 8) and 5′-TGAGCCCCTGCAGGCGCCCATGAAAAAGCCCGGC-3′(SEQ ID NO: 9), where the additionally introduced restriction sites ofHindIII and PstI sites are underlined.

The amplified DNA fragment of 545 bp, digested with HindIII and PstI,was inserted into the corresponding sites of pUC19 so that the azuringene was placed downstream of the lac promoter to yield an expressionplasmid pUC19-azuA. E. col JM109 was used as a host strain forexpression of the azurin gene. The recombinant E. coli strain wascultivated in 2YT medium containing 50 μg ml⁻¹ ampicillin, 0.1 mM IPTG;and 0.5 mM CuSO₄ for 16 h at 37° C. to produce azurin.

Site-directed Mutagenesis of the Azurin Gene

Site-directed mutagenesis of the azurin gene was performed using aQuickChange site-directed mutagenesis kit (Stratagene, La Jolla,Calif.). A single set of oligonucleotides was designed for each mutationas follows. For C112D: 5′-CAGTACATGTTCTTCGACACCTTCCCGGGCCAC-3′ (SEQ IDNO: 10) and 5′-TGGCCCGGGAAGGTGTCGAAGAACATGTACTGC-3′ (SEQ ID NO: 11); forM44K: 5′-CCTGCCGAAGAACGTCAAGGGCCACAACTGGG-3′ (SEQ ID NO: 12) and5′-CCCAGTTGTGGCCCTTGACGTTCTTCGGCAGG-3′(SEQ ID NO: 13); for M64E:5′-GGTCACCGACGGCGAGGCTTCCGGCCTGG-3′ (SEQ ID NO: 14) and5′-CCAGGCCGGAAGCCTCGCCGTCGGTGACC-3′(SEQ ID NO: 15). Mutations wereconfirmed by DNA sequencing.

Chimeric Mutants of Azurin

Amino acid residues of azurin that were deemed to be candidates forT-cell-epitopes were searched by GENETYX software (Software Development,Tokyo). Seven putative antigenic epitopes, EP1 to EP7, were found asfollows: EP1, I20TVDKS²⁵ (SEQ ID NO: 16); EP2, V49LSTAA⁵⁴ (SEQ ID NO:17); EP3, G58VVT⁶¹ (SEQ ID NO: 18); EP4, G63HASG⁶⁶(SEQ ID NO: 19); EP5,R79VIAH⁸³ (SEQ ID NO: 20); EP6, K85LIG⁸⁸(SEQ ID NO: 21); and EP7,M121KGTLT¹²⁶ (SEQ ID NO: 22).

Amino acid sequences of azurins from various microorganisms wereobtained from GenBank and aligned by GENETYX software to compare aminoacids around the putative T-cell epitope (EP) sites (FIG. 11( a)). EPsites, numbering 1-7, are shown with bars on the top of the sequences.PA, Pseudomonas aeruginosa PAO1 (SEQ ID NO: 23); AF, Alcaligenesfaecalis (SEQ ID NO: 24), AX, Achromobacter xylosoxidans ssp.denitrificans I (SEQ ID NO: 25); BB, Bordetella bronchiseptica (SEQ IDNO: 26), MJ, Methylomonas sp. J (SEQ ID NO: 27); NM, Neisseriameningitidis Z2491 (SEQ ID NO: 28), PF, Pseudomonas fiuorescen (SEQ IDNO: 29); PC, Pseudomonas chlororaphis (SEQ ID NO: 30), XE, Xylellafastidiosa 9a5c (SEQ ID NO: 31).

The replacements of the amino acids at the putative antigenic epitopesof P. aeruginosa with amino acids from other microbial azurins weredesigned to obtain chimeric azurins in which the antigenic epitopes werealtered. The chimeric mutants were constructed cumulatively bysite-directed mutagenesis and replacement of a BstEII restrictionfragment in the azurin gene using the following oligonucleotides: forT21 Qmutation within EP 1,5′-CAACACCAATGCCATCcagGTCGACAA GAGCTGCAAGC-3′(SEQ ID NO: 32)and 5′-AGCTCTTGTCGACctgGATGGCATTGGTGT TGAACTGC-3′ (SEQ IDNO: 33); for T126K mutation within EP7,5′-GAAGGGCACCCTGAag CTGAAGTGATGCGCG-3′ (SEQ ID NO: 34), and 5′-GCGCATCACTTCAG ctTCAGGGT GCCCTTCATC-3′(SEQ ID NO: 35); for T52K/A53S mutations withinEP2,5′-AACTGGGTACTGAGCAagtCCGCCGACATGCAGGGC-3′ (SEQ ID NO: 36) and5′-CTGCATGTCGGCGGactTGCTCAGTACCCAGTTG TG 3′ (SEQ ID NO: 37); forG58P/V591 mutations within EP3,5′-CCGCCGACATGCAGccCaTGGTCACCGACGGCATGGC-3′ (SEQ ID NO: 38) and 5′-GCCATGCCGTCGGTGACCAtGggCTGCATGTCGGCGG-3′ (SEQ ID NO: 39); for M591/V60A mutations withinEP3,5′-CATGCAGCCCA TcGcCACCGACGGCATGGC-3′ (SEQ ID NO: 40) and5′-CATGCCGTCGGTGgCgATGGG CTGCATGTCG-3′ (SEQ ID NO: 41); forS66AIG67AIH83F/K85P/L861 mutations within EP4, EP5, andEP6,5′-GTCACCGACGGCATGGCTgCCGcCCTGGACAAGGATTACCTGAAGCCCGACGACAGCCGTGTCATCGCCttCACccGaTcATCGGCTCGGGCGAGAAGG ACTCG-3′ (SEQ ID NO:42)and 5′GTCACCGAGTCCTTCTCGCCCGAGCCGATgAtCggGGTGaaGGCGATGACACGGCTGTCGTCGGGCTTCAGGTAATCCTTGTCCAGGgCGGcAGCCATGCCGTCG-3′ (SEQ ID NO: 43), in which a BstEII sitewas underlined, were used to replace BstEII fragments from the wt azuringene. Small letters in the oligonucleotides indicate the mutagenicnucleotides.

FIG. 11( b) shows wild type azurin and chimeric mutant azurins preparedusing the methods described above. S1 (SEQ ID NO. 45), S2 (SEQ ID NO.46), S3 (SEQ ID NO. 47), S4 (SEQ ID NO. 50), and S6 (SEQ ID NO. 51) wereconstructed in this order by site-directed mutagenesis cumulatively.WtS5 (SEQ ID NO. 52) and S3S5(SEQ ID NO. 48) were constructed byreplacement of the wt (SEQ ID NO. 44) BstEII fragments of wt azurin(wtS5) and S3 azurin (S3S5) with mutagenic BstEII fragment respectively.WtS5S4S6(SEQ ID NO. 53) and S3S5S4S6(SEQ ID NO. 49) were constructed bytwo rounds of site-directed mutagenesis using the wtS5 gene and the S3S5gene as template DNA respectively. Introduction of mutations wasconfirmed by DNA sequencing. Replaced amino acids are shown in bold. Thegenes were expressed in E. coli as described for wild type azurin above.No expression of S3S5S4S6 was observed.

Wild type and mutant azurins were purified from periplasmic fractions ofrecombinant E. coli cells using a Q-Sepharose FF column and a Superdex75 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according tothe method described by Kukimoto et al (1996). For the preparation ofapo-azurin, wild type azurin was treated with 0.1M MES buffer pH 6.0,containing 0.2M thiourea, 0.25M NaCl and 1 mM EDTA for 16 hr. Releasedcopper was removed by dialysis according to the method described by vanPouderroyen et al., Biochemistry, vol. 35, pp 1397-1407 (1996).

Example 20 Cytotoxic Activity of Azurin and Mutant Azurins

The wide type azurin, apo-azurin, C112D and M44KM64E mutants, and thechimeric mutants prepared in Example 19 were used in macrophagecytotoxicity assays. Macrophage isolation was as in Example 2.

Approximately 1×10⁵ cells per well were seeded into 96-well cultureplates in 200 μl of RPMI-1640 medium containing 10% FBS at 37° C. with5% CO₂. After overnight growth, the cells were washed with the samemedium, which was thee replaced with new medium containing azurin ormutant azurin. After 24 h treatment 10 μl of 5 mg mi⁴ MTT [3-(45-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide)] solution wasadded to the culture and incubated for 2.5 h at 37° C. MTT reaction wasterminated by the addition of 40 mM HCl in isopropanol. The MTT formazanformed was measured spectrophotometrically according to the methoddescribed by Mosmann, J. Immunol. Methods, vol. 65, pp 55-63 (1983).

The cytotoxicity of azurin and the mutant azurins is shown in FIGS. 12(a) and 12(b). FIG. 12( b) also shows the relative electron transferefficiency of the mutants expressed as a percentage of that of wild typeazurin. Here, the electron transfer efficiency between oxidized azurinand reduced cytochrome C₅₅₁ was measured by laser flash photolysis asdescribed by Cutruzzola et al., Journal of Inorganic Biochemistry 88;353-361, 2002.

Example 21 Apoptotic Activity of Azurin and Mutant Azurins

To determine the apoptosis rate induced by wild type azurin or azurinmutants, change in mitochondrial potential was measured by flowcytometry (Becton Dickinson, Inc., Franklin Lakes, N.J.) using anApoAlert mitochondrial membrane sensor kit (Clontech Laboratories, Inc.,Palo Alto, Calif., U.S.A.). Macrophage isolation was as in Example 2.

Approximately 1×10⁶ cells per well were seeded into six-well cultureplates in 2 ml of RPMI-1640 medium containing 10% FBS at 37° C. with 5%CO₂. After overnight growth, the cells were washed with the same medium,which was thee replaced with new medium containing azurin or mutantazurin. After 16 h treatment, the cells were stained with the MitoSensordye and analyzed by flow cytometry with a FL-1 filter according to themanufacturer's manuals.

FIG. 13 shows the apoptotic activity of azurin, apo-azurin, and theC112D and M44KM64E mutants towards macrophage cells. The apoptotic rate(%) is expressed as the fraction of the cell population that shiftedfrom the control population to a green fluorescing apoptotic population.

Example 22 Cytotoxic Activity of Rusticyanin, Apo-rusticyanin, andPseudoazurin

The wide type azurin was prepared as in Example 19. Rusticyanin fromThiobacillus ferrooxidans and pseudoazurin from Achromobactercycloclastes were prepared by hyperexpression of their genes and columnchromatographic fractionation as described for azurin (Yamada et al.2002; Goto et al. 2003). Apo-rusticyanin was prepared using the methoddescribed in Example 18. UISO-Mel-2 cells were obtained as in Example13.

Approximately 5×10³ cells per well were seeded into 96-well cultureplates in 200 μl of MEM medium containing 10% FBS at 37° C. with 5% CO₂.After overnight growth, the cells were washed with the same medium,which was then replaced with either buffer (PBS pH 7.4 or Tris-HCl pH5.0), crude sample containing rusticyanin in Tris-HCl pH5.0, orapo-rusticyanin in PBS pH 7.4. After 24 hr, a MTT assay was performed asdescribed in Example 19. The cytotoxicity of wild type azurin,rusticyanin, apo-rusticyanin and pseudoazurin is shown in FIG. 14.

Example 23 Cytotoxic Activity of Plastocyanin

The wide type azurin was prepared as in Example 18. Plastocyanin fromPhormidium laminosum was prepared by hyperexpression of its gene andcolumn chromatographic fractionation as described for azurin. Macrophageisolation was as in Example 2.

Approximately 1×10⁵ cells per well were seeded into 96-well cultureplates in 200 μl of RPMI-1640 medium containing 10% FBS at 37° C. with5% CO₂. After overnight growth, the cells were washed with the samemedium, which was thee replaced with new medium containing azurin ormutant azurin. After 24 hr treatment 10 μl of 5 mg/ml MTT[3-(45-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide)] solutionwas added to the culture and incubated for 2.5 hr at 37° C. MTT reactionwas terminated by the addition of 40 mM HCl in isopropanol. The MTTformazan formed was measured spectrophotometrically according to themethod described by Mosmann, J. Immunol. Methods, vol. 65, pp 55-63(1983). The cytotoxicity of wild type azurin and Plastocyanin is shownin FIG. 15.

1. A method of treating a condition related to resistance to cell death,comprising administering to a patient a pharmaceutical compositioncomprising azurin, to promote cell death in a cell demonstratingresistance to cell death wherein said azurin comprises the amino acidsequence of SEQ ID NO:
 1. 2. The method of claim 1, wherein the azurinbinds to tumor-suppressor protein p53.
 3. The method of claim 1 whereinthe condition related to resistance to cell death is selected from thegroup consisting of human melanoma, leukemia, breast cancer, ovariancancer, lung cancer, mesenchymal cancer, colon cancer and aerodigestivetract cancers.